Carbon fixation, or the conversion of CO2 to reduced forms amenable to cellular biochemistry, occurs by several metabolic pathways in diverse organisms. The most familiar of these is the Calvin Cycle (or “Calvin-Benson” cycle), which is present in cyanobacteria and their plastid derivatives (i.e., chloroplasts), as well as in proteobacteria. The Calvin cycle in these organisms utilizes the enzyme, ribulose-1,5-bisphosphate carboxylate/oxygenase (“Rubisco”). See, e.g., the world wide web at blc.Arizona.edu/courses/181gh/rick/photosynthesis/Calvin.html; Raven, et al. (1981) The Biology of Plants. 3rd Edition, Worth Publishers, Inc., NY, N.Y. Rubisco exists in at least two forms: Form I Rubisco, which is found in proteobacteria, cyanobacteria, and plastids; and Form II Rubisco, which is found in proteobacteria. Form I Rubisco is encoded by two genes encoding large and small subunits (rbcL and rbcS), and may exist as an octo-dimer composed of eight large subunits (rbcL) and eight small subunits (rbcS). Form II Rubisco is a dimeric form of the enzyme. Form II Rubisco has clear similarities to the large subunit of Form I Rubisco, and is encoded by a single gene, also referred to as rbcL. The evolutionary origin of the small subunit of Form I Rubisco remains uncertain; it is less highly conserved than the large subunit, and may have cryptic homology to a portion of the Form II protein.
All photosynthetic organisms catalyze the fixation of atmospheric CO2 by the bifunctional enzyme Rubisco. Significant variations in kinetic properties of this enzyme are found among various phylogenetic groups. Because of the abundance and fundamental importance of Rubisco, the enzyme has been extensively studied. Well over 1,000 different Rubisco homologues are available in the public literature and the crystal structure of Rubisco has been solved for several variants of the protein.
Rubisco contains two competing enzymatic activities: an oxygenase and a carboxylase activity. The oxygenation reaction catalyzed by Rubisco is considered a “wasteful” process because it competes with, and significantly reduces the net amount of carbon fixed by an organism. The Rubisco enzyme species encoded in various photosynthetic organisms have been selected by natural evolution to provide higher plants with a Rubisco enzyme that is substantially more efficient at carboxylation in the presence of atmospheric oxygen.
The creation of plants and other photosynthetic organisms having improved Rubisco biosynthetic pathways can provide increased yields of certain types of foodstuffs, enhanced biomass energy sources, and may alter the types and amounts of nutrients present in certain foodstuffs, among other desirable phenotypes. The development of technologies for effective biological fixation of CO2 on a global scale can mitigate the effects of atmospheric greenhouse gas emission. Cyanobacterial aquaculture (“cyanofarming”) offers one of the most productive solutions for global greenhouse gas control, as compared to other biological alternatives aimed at CO2 abatement technology for global use. However, it would be desirable to improve biomass productivity of cyanofarming by 10 to 20 fold over current production levels. Thus, a need exists for improved Rubisco enzymes.